INVESTIGATION OF THE ULTRAFINE STRUCTURE OF THE KIDNEY BY MEANS OF SCANNING ELECTRON MICROSCOPE

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1 THE KURUME MEDICAL JOURNAL 1975 Vol.22, No.3, P INVESTIGATION OF THE ULTRAFINE STRUCTURE OF THE KIDNEY BY MEANS OF SCANNING ELECTRON MICROSCOPE I. THE GLOMERULUS SHINSHI NODA Department of Urology, Kurume University School of Medicine, Kurume, 830, Japan (Received for publication September 25, 1975) The fine structures of the renal glomerulas, Bowman's capsule and the transitional area from Bowman's capsule to the proximal tubule have been studied after preparing with critical point method by field emission type scanning electron microscope. The structures of both the normal glomerular capillary wall and the basement membrane with three layers have been particularly examined. From the fine structure of the transitional area from Bowman's capsule to the proximal tubule, it has been suggested that this area has something to do with the flow of glomerular filtrate. Numerous investigations concerning the ultraf ine structure of the kidney have been performed by using transmission electron microscope. The results illustrate in detail normal and impaired renal structures of humans and animals. There is an impression about the presence of possible gap between the experimental results and their clinical contribution. The reason for this fact may consist in difficulty in making the experimental specimens. In addition, the same type of difficulty may exist in three-dimensional reconstruction of the renal structure from transmission electron micrographs. Recent application of scanning electron microscope for which specimen can be made more easily has rendered the three-dimensional survey of the micrographs possible, and its user is gradually increasing among clinicians. The present author who takes much interest in the ultrastructure of the kidney has accumulated experimental results of the renal structures by transmission electron microscope, and now he has also attempted to investigate the renal structures by field-emission type scanning electron microscope (resolving power, ð). This paper deals with the results in the latter category, and a part of the results has been preliminarily presented. MATERIALS AND METHODS The normal mature mice were used. First, the animals were perfused by physiological saline solution after canulation through the thoracic aorta. Next, fixation by perfusion was made with veronal acetate-buffered glutaraldehyde solution (ph 7.2). Then, the kidney was 135

2 136 NODA removed and cut to pieces. Further fixation was done with glutaraldehyde solution at 4 Ž for 2 hours. Washing was once more made with the same buffer solution. Post-fixation was performed with 1 % buffered osmium tetraoxide at 4 Ž for 2 hours. After dehydration with graded ethylalcohol series, the alcohol was displaced with acetic isoamyl by immersing for 2-3 hours. The tissue pieces were dried by critical point method and cut by frozen epoxy resin thinn section method. Spattering vapor coating of Au-Pd was made in the majority, and some of tissue pieces were coated with gold vapor by means of ion cleaner. Observation was made by using HFS-2 type scanning electron microscope. RESULTS AND DISCUSSION The nephron which is a structural and functional unit is composed of a glomerulus, a tubule (proximal, distal, including Henle's loop) and a collecting tube, and the number of the nephron was variously ranged from 1 to 4.5 millions by many authors. On the section surface, the cortex and medulla are evidently different in appearance, structure and function. Scanning electron micrograph of the cut surface demonstrates that the glomeruli exist within Bowman's capsules locating in the bulk of the tubuli which have been sectioned in various directions (Picture 1). When this glomerulus is observed after adequate magnification, glomerular capillaries are well expanded, and the octopus foot cells with branch- or finger-like processes are reported on those capillary surfaces by transmission electron microscopy (Picture 2). The foot cells are equivalent to the podocytes. Some thick primary processes are originating from one podocyte, and secondary or tertiary processes ramify in turn. Some of the primary processes connect immediately to the capillaries through the foot process. The podocytes in the mouse kidney arrange very regularly, and the foot processes branched off at a right angle from the primary processes of the adjoining podocytes interwine in a digital manner, the processes entering to the region beneath the thick process. The foot process is about mƒê wide, and the interspace between the processes is about ð in width. The interspace is named as a slit pore and is covered with the slit membrane, having thickness of about 70 ð, which can be revealed even by transmission electron microscope. The microvilli are few in number and can be recognized on the cellular surface. Besides the glomerulus, there is Bowman's capsule which is composed of the hyaline basement membrane made of mucopolysaccharide, the fibrous layer directly adjacent to the external interstitial tissue, and the internal parietal epithelium. The epithelial cell, flat and round, has microvilli, and each cell has respectively one nucleus at the centre of the cell and one cilium (Picture 3). The left part of the micrographic picture is equivalent to the part designated as a urinary pole connecting to the proximal convoluted tubule. The epithelial cells of the proximal convoluted tubule are characterized by the presence of peculiar brush border and the evident cubic form. When the region of the urinary pole is a little more magnified, the transitional portion from the glomerulus and Bowman's capsule to the proximal convoluted tubule becomes further evident as shown in Picture 4. The epithelial cells of the proximal convoluted tubule are conspicuously tall in height as compared with those of Bowman's capsule, and the free surface of

3 SCANNING EM OF RENAL GLOMERULUS 137 the former cells is characterized by the brush border compactly standing in a row. One interesting feature is that the topmost limit of the tubular cells having the brush border locates a little more above upwards than the funnelshaped portion of the proximal convoluted tubule (Pictures 3 and 4). Fig. 1 has been constructed by using Pictures 3 and 4, and indicates the microtopographical relationship between Bowman's capsule and the proximal tubule, suggesting the presence of certain mechanism regulating the orthodromic flow of glomerular filtrate. Fig. 1 Of course, the basal portion of Bowman's capsule and that of the proximal tubule are closely connecting to each other by the serial basement membrane, and the transitional region of the epithelia of both is characterized by the marginal folding. The glomerulus is a complicated capillary ball of which blood comes from the efferent arteriole and goes to the efferent arteriole, and it is subdivided into some lobules. The presence of both the anastomosis between intralobular capillaries and the anastomosis between adjacent lobular capillaries has been proved by stereographic construction of the serial preparations at the light microscopic level. The presence of such vascular anastomosis has also been demonstrated by the present author who has performed scanning electron microscopy of the vascular mold prepared with resin injection method. Thus, it is likely that the glomerulus is a network rather than a loop. The internal space of the glomerular capillaries is closely and compactly surrounded by the endothelial layer. There are numerous small pores on the endothelial cell covering the internal surface of the glomerular capillaries, and the pores are called as endothelial fenestration. The pores specific for the glomerular endothelium are about 600 ð in diameter and 70 ð in thickness (Pictures 5 and 6). There is no fenestration at the nuclear portion of the endothelial cells protruding into the internal space of the capillaries. The central knob at the centre of the endothelial fenestrations which has been recognized by transmission electron microscope cannot be reconfirmed by scanning electron microscope. The topographical relationship between the endothelial cell and the podocyte and in addition the situation of the basement membrane existing between them can be defined from Picture 6. The fine structure of the basement membrane has been first clarified by transmission electron microscopy. The basement membrane, as shown by Yamada 7?, is composed of three layers ; lamina densa with higher electron density at the central region and two layers (lamina rare externa et interna) holding the lamina densa between them. These are probably equivalent to three layer formation of the basement membrane by fine splitting as shown in Picture 6 (indicated by an arrow). Referring to these three layers there is a report that lamina densa in the middle portion

4 138 NODA has a function as the limit membrane for glomerular filtration, but further physiological investigation will be required as to this problem. ACKNOWLEDGEMENT I am indebted to Prof. K. Eto for his encouragement and criticism during the study. REFERENCES 1) FARQUHR, M.G.: Glomerular permeability. 1. Ferritin transfer across the normal glomerular capillary wall. J. Exptl. Med., 113, 47-66, ) HAMBURGER, J., RICHET, G. and GRUNFELD, J. P.: Organ physiology - structure and function of the kedney. W. B. Saunders Company, London ; ) MIYOSHI, M., FUJITA, T. and TOKUNAGA, J. A combined scanning and transmission electron microscope study in rats. Arch. Kist. jap., 33, , ) PEASE, D. C.: Fine structures of the kidney seen electron microscopy. J. Histochem. Cytochem., 3, , ) NODA, S., KAWADA, T. and ETO, K.: Scanning electron microscopic atlas 1. Kidney (1). Jap. J. din. Urol., 29, 53~-533, ) NODA, S., KAWADA, T. and ETO, K.: Scanning electron microscopic atlas 2. Kidney (2). Jap. J. din. Urol., 29, , ) YAMADA, E.: The fine structure of the renal glomerulus of the mouse. J. biophys. biochem. Cytol., 1, , EXPLANATION OF THE PICTURES (1) This is a weakly magnified figure of the cut surface of the renal cortex. Intricately running tubule and the glomerulus putting in Bowman's capsule are seen (indicated by an arrow) ( ~320). (2) In the sufficient magnification, there are podocytes (Po) covering the well expanded capillaries of the glomerulus. The podocyte demonstrates many secondary processes originating from the primary process. The process shows entanglement with the foot process (Fp) of the adjacent podocyte in a manner of three branches ( ~3, 000). Us : urinary space. Be : Bowman's capsule. (3) Picture shows Bowman's capsule wrapping the glomerulus, and the left side is the urinary pole shifting from Bowman's capsule (Bc) to the proximal tubule (Pt). The epithelial cell of Bowman's capsule is flat and round and presents the microvilli on its surface. In the middle of the epithelial cell there is a cilium ( ~1,400). (4) This is a magnified scanning electron micrograph. Topographic interrelationship among the glomerulus (G), Bowman's capsule and the proximal tubule (Pt) can be clearly observed ( ~3,100). Bb : brush border. (5) This shows a section surface of the glomerulus. The relationship between the opened capillary lumen and the podocyte covering the capillary is evident ( ~15, 000). Ca : capillary, Fp : foot process, Us : urinary space. (6) The internal surface of the capillary is covered by the endothelial cell, and the thinn portion of this cell presents a number of fenestrations. Three layers of the basement membrane can be seen between the endothelial cell and the podocyte (indicated by an arrow) ( ~31, 000). Ed : endotherial cell, Gcw : glomerular capillary wall.

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